This disclosure relates to biosensors for continuous monitoring of metabolites and proteins and to methods of manufacture thereof.
The development of biosensors for continuous monitoring of various analytes in the body of a living being is important because of its potential to provide an early indication of various body disorders and diseases. An important class of biosensors are electrochemical sensors that link enzymatic reactions to electroactive products. These sensors can be readily miniaturized and have enabled the detection of very small volumes of analytes in clinical or in home-use settings. For example, the development of miniaturized implantable sensors for continuous monitoring of glucose has revolutionized the management and care of diabetes mellitus.
In many other clinical situations, it is desirable to monitor the concentration of various metabolites developed and released into the bodies of living beings such as, for example, lactate, creatinine, creatine, glutamate, phosphate, cysteine, homocysteine, and the like. For example, a device that can measure lactate levels can be used in the detection of a number of diseases and conditions (e.g., to indicate muscle fatigue, shock, sepsis, kidney disorders, liver disorders, congestive heart failure amongst others). In some clinical situations, simultaneous monitoring of two or more metabolites is desirable. Because of the complex interrelationship between glucose and other metabolic analytes it is often desirable to simultaneously detect glucose, glutamate, lactate, oxygen, carbon dioxide, and the like. Simultaneous monitoring of glucose, lactate and/or oxygen levels in the brain provides a comprehensive picture of complementary energy supply to the brain in response to acute neuronal activation. Levels of glucose and glutamate in cerebrospinal fluid are important in the control of diseases such as meningitis.
Currently, most of the electrochemical sensors used for the specific detection of lactate, glucose, glutamate, and the like, employ analyte-specific enzymes, and are based on the Clark-type amperometric detection.
For example, first generation Clark-type glucose sensors employ the glucose oxidase enzyme (GOx), immobilized on top of a working electrode. This enzyme catalyses the oxidation of glucose to glucorolactone, as shown in reaction (1) below:
The generated hydrogen peroxide is amperometrically assessed on the surface of a working electrode according to reaction (2) below:
Clark-type bi-enzymatic sensors for detection of creatine employ two enzymes (namely creatinase and sacrosine oxidase) immobilized on top of the working electrode. First creatine is enzymatically converted by creatinase to sacrosine and urea (as shown in the reaction (3) below), the former of which is subsequently converted to glycine and hydrogen peroxide (H2O2) by the action of sacrosine oxidase enzyme (reaction 4).

Similar to glucose sensors, the generated hydrogen peroxide is amperometrically assessed on the surface of working electrode by relating the current to creatine concentration. As is evident from reactions (1) and (4), optimum sensor performance can only be attained when the ratio of the substrate (i.e., glucose or sacrosine) to co-substrate (i.e., oxygen) is less than 1. If this ratio is greater than or equal to 1, the lack of oxygen renders reactions 1 or 4 oxygen-limited. This results in inaccurate readings of glucose and creatine, respectively.
In the case of glucose, a 0.18 mM oxygen concentration in the subcutaneous tissue is substantially lower than the 5.6 mM of physiological glucose concentration (i.e., glucose/oxygen ratio of ca. ˜30). This leads to signal saturation at higher glucose concentrations. The onset of signal saturation is typically expressed as the apparent Michaelis-Menten constant, which defines the upper limit of the glucose range that the sensor can detect with enhanced confidence.
This issue has been addressed by the use of diffusion-limiting outer membranes that provide a greater permeability resistance to the larger sized substrate as opposed to the smaller sized co-substrate. As a result of this modification, semipermeable membranes based on NAFION®, polyurethane, cellulose acetate, epoxy resins, polyether-polyethersulfone copolymer membranes, and layer by layer (LBL) assembled polyelectrolytes and/or multivalent cations have been extensively investigated. However, in the use of semipermeable membranes it is desirable to have strict control over the thickness and uniformity of the outer membranes and this methodology comes at the expense of decreased sensitivity and increased sensor response time. Furthermore, the accumulation of exogenous reagents within these outer membranes (i.e., calcification, biofouling etc.) lead to sensor drift and therefore to their eventual failure.
In order to overcome this problem, an additional oxygen reservoir is provided within the outer membrane by incorporating oxygen-absorbing zeolites. Similarly, an oxygen reservoir (e.g., fluorocarbons, mineral oils and myoglobin) can be employed within the glucose oxidase enzyme layer to compensate for the decreased oxygen.
The eventual fabrication of multiple enzymatic Clark type sensors adjacent to each other necessitates a site specific deposition technique along with provisions to avoid crosstalk from one sensor element to another from the outward diffusing hydrogen peroxide. In all of the various methodologies described above, these provisions have not been implemented. Moreover, the growth of oxygen reservoir may be difficult to be implemented at will on a specific sensing element and not on another, since patterning of biological containing entities is at best challenging.
In another variation, second- and third-generation Clark type biosensors employ redox mediators and direct ‘wiring’ of enzymes to electrodes in an attempt to minimize the effects of oxygen concentration on the measurement of the analyte. In the case of mediators, their toxicity and biocompatibility along with the possibility to leach out from the device to the surrounding tissue present a major problem. Direct wiring of enzymes to electrodes minimizes these limitations, although adds unwanted complexities and higher expense.
The significant imbalance of glucose (as well as other analytes) to oxygen has prompted researchers to simultaneously measure substrate and co-substrate concentrations in order to account for co-substrate induced variations. Although this approach has its merits, this methodology is also prone to interferences from exogenous agents that render such calibration challenging.
Interferences from endogenous species (other than the primary substrate) generally originate from the fact that these species oxidize at the same potential as hydrogen peroxide. For example, in voltages of about 0.6 to about 0.7 volts (V) many endogenous species such as bilirubin, creatinine, L-cystine, glycine, ascorbic acid (AA), acetaminophen (AP), uric acid (UA), and the like, also get oxidized (leading to an erroneous electrochemical signal).
In order to increase confidence in sensing accuracy it is desirable to actively account for the signal generated by the endogenous species. At present not many methodologies have been developed to actively account for this. Anionic charged membranes (e.g., NAFION®, polyester sulfonic acid, cellulose acetate, and the like) have shown to exclude interferences from anionic species like ascorbic acid, uric acid, and so on, based on the principle of charge repulsion. These methods, however, inevitably impede permeation of negatively charged analyte species (e.g., lactate, pyruvate, glutamate, and the like), and render their detection challenging. In addition, the large response time associated with the diffusion of analytes through these membranes require long equilibration times in order to attain steady state performance between the inner and outer membrane, which is an additional drawback.
Another approach to eliminate interference signals from endogenous species has been the use of inner, ultra-thin, electropolymerized films between the working electrode and the enzyme layer. These films have been seen to partially screen analytes and analyte sensors from the interference agents. However, while these electropolymerized films minimize the contributions to signal from the endogenous species, they do not completely eliminate them.
In another approach, secondary enzymes (for example, ascorbate oxidase, which converts ascorbic acid to dehydroascorbate and water) have been incorporated in the outer membrane of the sensor to eliminate the particular species from reaching the electrode surface and contributing to the amperometric current. These secondary enzymes, do however, require oxygen as co-substrate and therefore have the potential of depleting the sensor of oxygen, which can negatively impact the operation of the primary enzyme.
Another major problem with the current state of the art biosensors is the unwanted production of byproducts as a result of enzymatic and/or electrochemical reactions. These unwanted byproducts tend to build up and/or adsorb on the surface of the working electrode leading to loss of function of the working electrodes. In some cases, the presence of these unwanted byproducts could also hinder the diffusion of analytes towards the working electrode as well as inhibit the progress of enzymatic reactions. For example, a lactate biosensor employing lactate oxidase works on the basis of the following reaction (5) below:

The generated hydrogen peroxide is amperometrically assessed on the surface of a working electrode by applying a positive potential, as shown above in reaction 2. However, because of the application of positive bias onto the working electrode, the negatively charged pyruvate (generated in reaction 5) tends to electrostatically adsorb on its surface leading to (i) taint the working electrode and subsequent loss of sensor sensitivity and (ii) inhibition of the reaction of lactate oxidase (reaction 5) with subsequent erroneous readings. To this end, higher applied potentials, double pulsed amperometry or pulsed amperometric detection have been the common strategies to renew the surface of the working electrode even though such techniques are complex to be applied for miniaturized sensors and implantable sensors with miniaturized driving electronics.
Because of its role to every metabolic activity of the body, the level of glucose is expected to vary following trauma, fever, exercise and/or another physical activities. Implantable glucose sensors can be made more reliable only when one takes into consideration the local and physiological variations in various metabolites that are in relation to glucose. These metabolites include oxygen, lactate and a number of proteins that take part in the glycolysis cycle. Some of these proteins also accelerate the generation as well as breakdown of various other body metabolites. While the levels of these body metabolites are indicative of the state of the body, in some case the activity of the proteins/enzymes itself is an indication of the state of the body. For example, the enzyme glutamic oxaloacetic transaminase (GOT), when found in elevated levels is an indicator of damage to liver (caused by viral hepatitis, heart attack, inflammation, alcohol abuse, and the like), as well as to pancreas, kidney, muscles and red blood cells due to injury.
A number of reports have also been disclosed for the fabrication and detection of glucose in conjunction with other metabolites. Similar to the glucose sensors, these more complex devices utilize outer semi-permeable membranes in order to account for interferences from oxygen and other endogenous species. In the detection of these metabolites it is generally desirable to perform a small number of sequential reactions in order to generate an electroactive species for electrochemical detection. Detection of proteins (enzymes as well as antibodies) is much more difficult and generally utilize enzyme linked immunosorbed assay (ELISA) methodologies. These methodologies are extremely difficult to be performed in-vivo and in a continuous manner. Developing methodologies to perform continuous protein detection in-vivo are therefore desirable for patients with a number of disorders.